Excision of 5-hydroxymethylcytosine by DEMETER DNA glycosylase from Arabidopsis

Abstract

In plants and animals, 5-methylcytosine (5mC) serves as an epigenetic mark to repress gene expression and plays important roles in gene imprinting and transposon silencing. The mammalian genome also contains 5-hydroxymethylcytosine (5hmC), formed by TET family-mediated oxidation of 5mC, which is referred to as the 6th base. 5hmC is abundant in mouse Purkinje neurons and embryonic stem cells, and regarded as an important intermediate during active DNA demethylation in mammals. However, it is not clearly demonstrated that 5hmC is present in plants. In Arabidopsis, DEMETER (DME) DNA glycosylase efficiently removes 5mC, which results in DNA demethylation and transcriptional activation of target genes. Here I showed that 5hmC was also excised by DME in vitro, raising the possibility that 5hmC may exist in plants and serve as an important intermediate for DNA demethylation. I performed a two dimensional thin layer chromatography (2D-TLC) analysis to verify whether 5hmC is present in Arabidopsis. I detected no or very little, if any, 5hmC from the Arabidopsis genome, suggesting that it is very unlikely for plants to have 5hmC. In addition, I searched for DME mutants with altered substrate specificity. A DME mutant protein, Q1362R, was capable of excising mismatched thymine more readily than 5mC. Our findings suggest that the region upstream of the Fe-S cluster is crucial for shaping the recognition pocket of DME, providing the substrate specificity for this family of enzymes.